CN114921403B - Full-automatic mouse bone marrow flushing device and bone marrow cell extraction method thereof - Google Patents

Abstract

The invention discloses a full-automatic mouse bone marrow flushing device and a bone marrow cell extraction method thereof, wherein the device comprises a PCL controller, a power management module, a micro pump, an electromagnetic valve group, a cleaning liquid storage tank, an electric shearing assembly, a first electric screw rod assembly and a second electric screw rod assembly, wherein the PCL controller is used for programming and controlling the electric shearing assembly to automatically shear epiphysis operation, the first electric screw rod assembly and the second electric screw rod assembly are mutually matched to run so as to complete epiphysis shearing, femur and/or tibia movement, and the collection cavity and the collection groove are automatically flushed, so that convenience operation is provided for reducing pollution probability and simplifying experimental steps. The invention uses the spherical filter cover to cut epiphysis and filter liquid, which is beneficial to the subsequent culture of bone marrow cells. The method has the advantages of simple and convenient operation, high efficiency, difficult pollution to cells, difficult damage to cells, capability of obtaining a large number of mouse bone marrow cells with better activity and good application prospect.

Description

Full-automatic mouse bone marrow flushing device and bone marrow cell extraction method thereof

Technical Field

The invention relates to the technical field of bone marrow extraction equipment in biological experiments, in particular to a full-automatic mouse bone marrow flushing device and a bone marrow cell extraction method thereof.

Background

Free mouse bone marrow cells are an essential procedure in cytological studies. Bone marrow cells include stromal cells and hematopoietic cells, and hematopoietic stem cells are adult stem cells in the blood system that have long-term self-renewal capacity and the potential to differentiate into various mature blood cells, so that the isolation of bone marrow cells is the basis for scientific research of blood system diseases and immune system diseases. Meanwhile, the bone marrow mesenchymal stem cells have the potential of multidirectional differentiation, and become a research hotspot of multiple subjects such as cardiovascular, nervous, endocrine, oral cavity and the like. How to efficiently isolate bone marrow cells has become an important issue for many researchers.

In reality, the adoption of model biological development research is particularly necessary because of the limitation of normal people and the sample acquisition way of patients. Wherein mice are the most widely studied model organisms using the most genetic background. Whether stem cell research or blood disease field research, bone marrow extraction from the bones of the extremities of mice is a frequently involved experimental link in scientific research.

Currently, there are two conventional bone marrow extraction methods: firstly, the injector method is to cut off two epiphyses of a tibia or a femur of a mouse to expose a bone marrow cavity, and then wash out bone marrow with a cell culture medium, but the method can lead to a great deal of bone marrow loss, has longer extraction time and low efficiency, reduces the activity of cells, uses more instruments such as forceps and scissors, causes pollution of the cells and seriously affects the research efficiency; secondly, the bone marrow direct puncture method is to puncture the lower limb bone by a puncture needle and suck the bone marrow, and because the limbs of the mice are short and the bone marrow amount is small, the puncture operation is difficult to carry out, even if the operation is still carried out for a plurality of times, the process is tedious, and the punctured bone marrow cells are seriously damaged and have rare quantity.

In the prior published paper, on the 4 th month and 2 nd year of 2011, the Babyshikimi et al published "reverse anatomical free femur/tibia rapid preparation of mouse bone marrow cells" on the 15 th edition in China tissue engineering research and clinical rehabilitation ", the authors adopt reverse muscle trend to free the muscle by shearing force of scissors, the muscle can be completely stripped by proper force, particularly the knee joint part adopts a reverse turning action opposite to the movement of the joint, so that the femur and the tibia metaphyseal are clean after being free, and the reverse anatomical method can solve the problems of unclean muscle, broken bone, pollution and the like to a certain extent, but also has a plurality of problems in the conventional bone marrow extraction method.

In the prior published patent document, 2021, 4 and 6 days discloses a marrow extraction device (application publication number: CN 112603387A) for blood medicine, and the technical scheme disclosed in the patent document is also a device for extracting marrow, and can simplify the operation steps of workers to a certain extent, but has no reference value for applicability and reference of extracting marrow from bones of limbs of mice.

The technical scheme disclosed in 2022, month 10 and 22 (issued bulletin number: CN 214437150U) comprises a centrifugal collecting tube, a centrifugal container and a filtering mechanism, wherein the centrifugal container is fixed in the centrifugal collecting tube, the filtering mechanism is fixed in the centrifugal collecting tube and is arranged around the outer side of the centrifugal container to collect bone marrow cells after being centrifugally filtered from the centrifugal container, the technical problems that the bone marrow cell extraction method is difficult to extract bone marrow cells and is not pure are solved, but the problem that the bone marrow collection amount is small in the conventional bone marrow extraction method is solved, but other problems in the conventional bone marrow extraction method are not recorded in related technical schemes, so the technical scheme of the patent can not meet the bone marrow extraction requirement of a femur or a tibia far away.

A bone marrow cell preparation device (grant bulletin number: CN 215162779U) disclosed in 12/14/2021, which comprises a bone scaffold, a bone marrow cell collection vessel, a flushing fluid vessel, a peristaltic pump, a first connecting tube, a second connecting tube and an ultrasonic instrument; the bone bracket is used for fixing the broken bone above the collecting port of the bone marrow cell collecting vessel in a way of downward section, one end of the first connecting pipe is inserted into the flushing fluid vessel, the other end of the first connecting pipe is connected with the liquid inlet of the peristaltic pump, one end of the second connecting pipe is connected with the liquid outlet of the peristaltic pump, and the other end of the second connecting pipe is connected with the injection needle; the ultrasonic instrument is provided with an ultrasonic probe, under the working condition of cell preparation, cell flushing liquid is contained in the flushing liquid vessel, the injection needle is inserted into a broken bone fixed on the bone bracket, and the ultrasonic probe is inserted into the section of the broken bone. According to the technical scheme, only partial manual operation can be replaced, the degree of automation is low, meanwhile, the particle vibration principle is adopted in the patent, namely, cavitation, acceleration and direct flow action of ultrasonic waves in liquid are utilized to directly and indirectly act on the liquid and objects, so that cells are dispersed, emulsified and peeled off, and further bone marrow cells are prepared.

Disclosure of Invention

Aiming at the problems that the existing method for extracting the mouse bone marrow cells in the background technology is difficult to control the washing force during operation, is easy to damage cells, is tedious in extraction of the mouse bone marrow cells for batch experiments, is time-consuming and labor-consuming, is difficult to operate by new laboratory workers, is often matched with a plurality of instruments, is easy to cause cell pollution, and further affects the subsequent cell culture, the invention provides a full-automatic mouse bone marrow washing device and a bone marrow cell extraction method based on the traditional method for washing the mouse bone marrow by using a 1mL injector.

In order to achieve the above purpose, the invention adopts the following technical scheme: a full-automatic mouse bone marrow flushing device comprises an outer shell, wherein lifting handles are respectively arranged on two opposite side surfaces of the outer shell, a cover plate is arranged on the back surface of the outer shell, a PCL controller and a power button are respectively arranged on the top surface of the outer shell, a lithium battery, a power management module, a micropump, an electromagnetic valve group and a cleaning fluid storage tank are respectively arranged in the outer shell, the output end of the power management module is electrically connected with the lithium battery and charges the lithium battery, the output end of the power management module is electrically connected with the PCL controller and supplies power to the PCL controller, a power button is connected in series between the PCL controller and the power management module, the power button is used for controlling the circuit on-off of the PCL controller, a charging interface is arranged on the power management module, the charging interface is used for externally connecting the power adapter to charge the lithium battery through the power management module, the micropump and the electromagnetic valve group are respectively electrically connected with the output end of the PCL controller, the micropump and the electromagnetic valve group are controlled through programming of the PCL controller, the electromagnetic valve group, the outlet of the cleaning fluid storage tank is communicated with the inlet of the micropump through a pipeline, the outlet of the electromagnetic valve group is also fixedly connected with the corresponding collecting needle through a plurality of collecting needle grooves on a movable needle, a collecting needle is arranged on a movable needle, a collecting needle is fixedly arranged on the bottom of a movable needle, a collecting needle is fixedly connected to a collecting needle, a collecting needle is fixedly arranged on the bottom of the collecting needle, and a movable needle is fixedly connected to the collecting needle, the base support is installed in the shell, the bottom of the collecting tank is connected with the supporting frame through a telescopic hose, a collecting box is slidably arranged on the supporting frame corresponding to the outlet of the telescopic hose, a turnover panel is hinged to the front face of the shell, a movable plate is slidably arranged on the inner side face of the turnover panel, the movable plate is powered by a second electric screw rod assembly to move up and down, a pair of clamping pieces and electric shearing assemblies are arranged on the movable plate corresponding to each flushing needle, each clamping piece and each electric shearing assembly are installed on the movable plate through a mounting bottom plate, each pair of clamping pieces are jointly used for clamping thighbone or tibia of the same mouse, corresponding two electric shearing assemblies are used for shearing epiphysis arranged at two ends of the thighbone or tibia, and the first electric screw rod assembly, the second electric screw rod assembly and the electric shearing assemblies are controlled to respectively operate and cooperate to complete shearing of the epiphysis performed by executing editing instructions of a PCL controller, and movement of the thighbone and/or tibia, and movement of the collecting cavity and the collecting tank are controlled by the electric shearing assemblies.

As a further step of the above embodiment, the first electric screw rod assembly includes a first servo motor installed on the support frame, a first screw rod is provided at an output end of the first servo motor, the other end of the first screw rod is rotatably provided on the support frame, a first screw plate is installed on the collecting tank corresponding to the first screw rod, sliding rails are respectively provided at four corners of the collecting tank corresponding to the support frame, and the first servo motor is electrically connected with an output end of the PCL controller and controls forward or reverse operation of the first servo motor through an editing instruction of the PCL controller.

As a further step of the above embodiment, the second electric screw assembly includes two second servomotors mounted on the turnover panel, each second servomotor is provided with a second screw on its output end, the other end of the second screw is rotatably mounted on the turnover panel, at least two second screw plates are mounted on the movable plate corresponding to each second screw, two guide rail grooves are provided on the turnover panel, two guide rail grooves are provided on the movable plate corresponding to the two guide rail grooves, the second servomotors are electrically connected with the output end of the PCL controller, and the forward or reverse operation of the second servomotors is controlled by the edit instruction of the PCL controller.

As a further step of the above embodiment, each electric shear assembly comprises an arc-shaped blade hinged on a mounting bottom plate, and a blade slot corresponding to the arc-shaped blade and fixedly connected on the mounting bottom plate, wherein a plurality of arc-shaped blades positioned on the same level are all hinged on a transmission rod through hinged connection sheets, at least two limit sliding sleeves are installed on the movable plate corresponding to each transmission rod, racks are fixedly connected on the transmission rods, a transmission shaft is rotatably arranged on the movable plate, transmission gears meshed with the racks are respectively arranged at two ends of the transmission shaft, a driving motor is further installed on the movable plate, meshed conical gears are respectively installed on an output shaft and the transmission shaft of the driving motor, and the driving motor is electrically connected with an output end of a PCL controller and controls forward or reverse running of the driving motor through editing instructions of the PCL controller.

Further to the above embodiment, the end of the flushing needle is communicated with a hollow ball, and a plurality of flushing holes are formed in the hollow ball.

As a further development of the above embodiment, a spherical filter housing is provided in each of the collecting chambers, the spherical filter housing being a mesh screen for collecting the sheared epiphysis, thereby preventing the epiphysis from being blocked into the flow-through tube or into the collecting tank.

Further to the above embodiment, a liquid filling port is further provided on the top surface of the outer case, a cap is mounted on the liquid filling port, and a liquid filling connection pipe communicating with the liquid filling port is provided on the cleaning liquid storage tank.

As a further development of the above embodiment, a pull handle and a push latch for temporarily locking the flip panel to the outer housing are provided on the outer side of the flip panel.

As a further step of the above embodiment, a support platform for supporting the flipped panel after flipping is also mounted on the front surface of the outer case.

The method for extracting the bone marrow cells by adopting the full-automatic mouse bone marrow flushing device in the technical scheme comprises the following steps:

step 1: opening a turnover panel to enable the turnover panel to be turned over to a supporting platform, placing thighbone and/or tibia of a stripped mouse on each pair of clamping pieces in sequence by using forceps, enabling epiphyses at two ends of each thighbone or tibia to be positioned between an arc-shaped blade and a cutting edge groove, and closing the turnover panel on the outer shell;

step 2: pressing a power button to start the device and placing the device on an ultra-clean workbench;

step 3: the driving motor starts and drives the arc-shaped blade to rotate according to the editing instruction of the PCL controller to cut off epiphysis at two ends of each femur or tibia, and the cutting operation instruction is completed;

step 4: the two second servo motors start the common driving movable plate to move upwards according to the editing instruction of the PCL controller, so that the flushing needle is inserted into the femur or tibia to finish the alignment access operation instruction;

step 5: firstly, an electromagnetic valve group turns on a pipeline switch provided with femur or tibia according to an editing instruction of a PCL controller, then a micro pump is started according to the editing instruction of the PCL controller, hanks buffer solution in a cleaning solution storage tank is pumped into a marrow cavity, then two second servo motors start to jointly drive a movable plate to move up and down circularly according to the editing instruction of the PCL controller, meanwhile, a first servo motor starts to enable a collecting tank and the collecting cavity to move up and down circularly in cooperation with the movable plate according to the editing instruction of the PCL controller, mouse marrow suspension sequentially enters the collecting cavity and the collecting box through a spherical filter cover, and part of air enters the collecting tank in the telescopic process of a telescopic hose in the whole up and down circulating moving process, so that the air entering the collecting tank enters the cell flushing liquid in the collecting box along the telescopic hose to play a role of dropper flushing;

step 6: after the step is completed, the micropump is closed according to the editing instruction of the PCL controller, then the electromagnetic valve group closes all flushing channel switches according to the editing instruction of the PCL controller, and finally the two second servo motors start the common driving movable plate to move downwards according to the editing instruction of the PCL controller so that the flushing needle is completely separated from the bone marrow cavity of the femur or tibia, and the flushing operation instruction is completed;

step 7: the ultra-clean workbench is opened, the device is taken out, the collecting box is pulled out, and the cell flushing liquid is uniformly pumped into the container by using the dropper for standby.

Compared with the prior art, the invention has the following advantages: according to the invention, the PCL controller is used for programming the starting time and sequence of the second servo motor, the driving motor, the micropump, the electromagnetic valve group and the first servo motor, so that the automatic epiphysis shearing operation of the electric shearing assembly is controlled, the first electric screw rod assembly and the second electric screw rod assembly are mutually matched to operate to complete epiphysis shearing, femur and/or tibia moving, and the collecting cavity and the collecting groove are automatically flushed, thereby providing convenience operation for reducing pollution probability and simplifying experimental steps. The invention uses the spherical filter cover to cut epiphysis and filter liquid, which is beneficial to the subsequent culture of bone marrow cells. The invention introduces ultraviolet irradiation sterilizing equipment to ensure the safety and the cleanliness of the instrument and provides a constant temperature and humidity environment for preventing cells from being influenced by the temperature and humidity of gas. The method has the advantages of simple and convenient operation, high efficiency, difficult pollution to cells, difficult damage to cells, capability of obtaining a large number of mouse bone marrow cells with better activity and good application prospect.

The device and the method of the invention simply and efficiently extract the bone marrow cells of the mice on the premise of ensuring the cell activity. The invention provides a novel, simple, efficient and practical device for the related research of hematopoietic cells, and overcomes the defects of difficult control of flushing force, easy damage to bone marrow cell activity, easy pollution, large difficulty in extraction of the rat bone marrow cells for batch experiments, complex operation and the like in the traditional flushing method. The extraction efficiency of hematopoietic stem cells in the related basic research of the bone marrow transplantation of the mice can be improved, the experimental time is saved, the cost is saved, the experimental progress is advanced, and the experimental success rate is improved.

Drawings

FIG. 1 is a perspective view of the southeast axial side of an embodiment of the present invention;

FIG. 2 is a perspective view of the northwest axis of an embodiment of the present invention;

FIG. 3 is a schematic view of an embodiment of the invention illustrating an open state of a flip panel;

FIG. 4 is an internal block diagram of the southeast axial side direction of an embodiment of the present invention;

FIG. 5 is an assembled view of the southeast axial side direction according to the embodiment of the present invention;

FIG. 6 is an assembled view of the northeast axis of the present invention;

FIG. 7 is an assembled view of an electric shears assembly according to an embodiment of the present invention;

FIG. 8 is a sliding fit assembly view of the flip panel and the movable plate according to the embodiment of the present invention;

FIG. 9 is a schematic view of an electric shears assembly according to an embodiment of the present invention;

FIG. 10 is an enlarged view in section of a flush needle in an embodiment of the invention;

FIG. 11 is a schematic perspective view of a spherical filter housing according to an embodiment of the invention;

FIG. 12 is a circuit connection block diagram of an embodiment of the present invention;

FIG. 13 is a schematic circuit diagram of a power management module and an STM32RCT6 singlechip in an embodiment of the invention;

FIG. 14 is a schematic diagram of a reference for use in accordance with an embodiment of the present invention;

FIG. 15 is a graph comparing the effects of a femoral and tibial bone marrow cavity of a mouse before and after rinsing using a conventional manual rinsing and the device of the present invention, wherein FIG. a is a graph showing the effects of a conventional manual rinsing before and after rinsing, and FIG. b is a graph showing the effects of a device of the present invention before and after rinsing;

FIG. 16 is an electron micrograph of the bone marrow hematopoietic stem cell population obtained using the apparatus and the extraction method of the present invention and the conventional manual extraction method, wherein FIG. c is an electron micrograph of the bone marrow hematopoietic stem cell population obtained using the apparatus of the present invention and FIG. d is an electron micrograph of the bone marrow hematopoietic stem cell population obtained by conventional manual extraction method.

In the figure: the device comprises a shell 1, a liquid injection port 101, a cover plate 102, a supporting platform 103, a lifting handle 2, a cap 3, a turnover panel 4, a collection box 5, a PCL controller 6, a charging port 7, a power button 8, a pressing lock catch 9, a handle 10, a movable plate 11, a second servo motor 12, a second screw rod 13, a mounting base plate 14, a guide rail groove 15, an electric scissor assembly 16, a cutting edge groove 1601, an arc blade 1602, a transmission rod 1603, a rack 1604, a transmission gear 1605, a transmission shaft 1606, a driving motor 1607, a hinged connection sheet 1608, a base bracket 17, a support frame 18, a lithium battery 19, a power management module 20, a micropump 21, an electromagnetic valve bank 22, a cleaning liquid storage tank 23, a liquid injection connection pipe 24, a buffer cavity 25, a flushing needle 26, a hollow ball 2601, a flushing hole 2602, a collection cavity 27, a spherical filter cover 28, a flow tube 29, a fixed plate 31, a collection groove 30, a telescopic hose 32, a slide rail 33, a first screw rod 34, a first servo motor 35, a clip 36, a guide rail 37, a limit slide sleeve 38, a screw hole plate 39, a second screw hole plate 40.

Detailed Description

In order to further illustrate the technical scheme of the invention, the invention is further illustrated by the following examples.

As shown in fig. 1 to 12, a full-automatic mouse bone marrow flushing device comprises an outer shell 1, handles 2 are respectively arranged on two opposite side surfaces of the outer shell 1, a cover plate 102 is arranged on the back surface of the outer shell, a PCL controller 6 and a power button 8 are respectively arranged on the top surface of the outer shell 1, a lithium battery 19, a power management module 20, a micropump 21, an electromagnetic valve bank 22 and a cleaning liquid storage tank 23 are respectively arranged in the outer shell 1, the output end of the power management module 20 is electrically connected with the lithium battery 19 and charges the lithium battery 19, the output end of the power management module is electrically connected with the PCL controller 6 and supplies power to the PCL controller 6, a power button 8 is connected in series between the PCL controller 6 and the power management module 20, the power button 8 is used for controlling the circuit on-off of the PCL controller 6, a charging interface 7 is arranged on the power management module 20, the charging interface 7 is used for externally connecting a power adapter to charge the lithium battery 19 through the power management module 20, the micro pump 21 and the electromagnetic valve group 22 are respectively and electrically connected with the output end of the PCL controller 6, the micro pump 21 and the electromagnetic valve group 22 are controlled to operate through programming of the PCL controller 6, the outlet of the cleaning liquid storage tank 23 is communicated with the inlet of the micro pump 21 through a pipeline, the outlet of the micro pump 21 is communicated with the inlet of the electromagnetic valve group 22 through a pipeline, a plurality of outlets of the electromagnetic valve group 22 are respectively connected with a plurality of buffer cavity pipes 25 through pipelines, each buffer cavity pipe 25 is provided with a flushing needle 26, the end part of the flushing needle 26 is communicated with a hollow sphere 2601, a plurality of flushing holes 2602 are formed in the hollow sphere 2601, a collecting cavity 27 is also arranged in the outer shell 1 corresponding to each flushing needle 26, the bottom of each collecting cavity 27 is communicated with a flow pipe 29, the flow pipes 29 are fixedly connected to a collecting tank 30 through a fixing plate 31, the collecting tank 30 is movably arranged on a supporting frame 18 and is powered by a first electric screw rod assembly to move up and down, a spherical filter cover 28 is arranged in each collecting cavity 27, the spherical filter cover 28 is a 300-mesh screen for collecting sheared epiphysis, so that the epiphysis can be prevented from entering the flow pipes 29 to be blocked or entering the collecting tank 30, the supporting frame 18 is arranged on a base bracket 17, the base bracket 17 is arranged in an outer shell 1, the bottom of the collecting tank 30 is connected with the supporting frame 18 through a telescopic hose 32, a collecting box 5 is slidably arranged on the supporting frame 18 corresponding to the outlet of the telescopic hose 32, a turnover panel 4 is hinged on the front surface of the outer shell 1, a handle 10 and a pressing lock catch 9 are arranged on the outer side surface of the turnover panel 4, the pressing lock catch 9 is used for temporarily locking the turnover panel 4 on the outer shell 1, a supporting platform 103 is also arranged on the front surface of the outer shell 1, the supporting platform 103 is used for supporting the turnover panel 4 after turnover, a movable plate 11 is slidably arranged on the inner side surface of the turnover panel 4, the movable plate 11 is powered by a second electric screw rod assembly to move up and down, a pair of clamping pieces 36 and electric shearing assemblies 16 are arranged on the movable plate 11 corresponding to each flushing needle 26, each clamping piece 36 and each electric shearing assembly 16 is arranged on the movable plate 11 through a mounting bottom plate 14, each pair of clamping pieces 36 are jointly used for clamping the femur or tibia of the same mouse, and the two corresponding electric shearing assemblies 16 shear the bone at two ends of the femur or tibia, the first electric screw rod assembly, the second electric screw rod assembly and the electric shear assembly 16 respectively control the operation and coordination of the first electric screw rod assembly, the second electric screw rod assembly and the electric shear assembly by executing the editing command of the PCL controller 6 to complete the shearing of the epiphysis, the movement of the femur and/or the tibia, the movement of the collecting cavity 27 and the collecting groove 30. A liquid filling port 101 is further formed in the top surface of the outer casing 1, a cap 3 is mounted on the liquid filling port 101, and a liquid filling connection pipe 24 communicated with the liquid filling port 101 is arranged on the cleaning liquid storage tank 23.

As a further preferred embodiment of the foregoing technical solution, the first electric screw assembly includes a first servo motor 35 mounted on the support frame 18, a first screw rod 34 is disposed at an output end of the first servo motor 35, the other end of the first screw rod 34 is rotatably disposed on the support frame 18, a first screw plate 39 is mounted on the collecting tank 30 corresponding to the first screw rod 34, sliding rails 33 are respectively disposed at four corners of the support frame 18 corresponding to the collecting tank 30, and the first servo motor 35 is electrically connected with an output end of the PCL controller 6 and controls forward or reverse operation of the first servo motor 35 through an edit instruction of the PCL controller 6.

As a further preferred embodiment of the foregoing technical solution, the second electric screw assembly includes two second servomotors 12 mounted on the turnover panel 4, each second servomotor 12 is provided with a second screw 13 at an output end thereof, the other end of the second screw 13 is rotatably disposed on the turnover panel 4, at least two second screw plates 40 are mounted on the movable plate 11 corresponding to each second screw 13, two guide rail grooves 15 are disposed on the turnover panel 4, guide rails 37 are disposed on the movable plate 11 corresponding to the two guide rail grooves 15, and the second servomotors 12 are electrically connected with an output end of the PCL controller 6 and control forward or reverse operation of the second servomotors 12 through editing instructions of the PCL controller 6.

As a further preferred embodiment of the foregoing technical solution, each of the electric shears assemblies 16 includes an arc blade 1602 hinged to the mounting base plate 14, and a blade slot 1601 fixedly connected to the mounting base plate 14 corresponding to the arc blade 1602, where a plurality of arc blades 1602 located on the same level are all hinged to a transmission rod 1603 through hinge connection pieces 1608, at least two limit sliding sleeves 38 are installed on the movable plate 11 corresponding to each transmission rod 1603, racks 1604 are fixedly connected to the transmission rods 1603, a transmission shaft 1606 is rotatably provided on the movable plate 11, transmission gears 1605 meshed with the racks 1604 are respectively provided at two ends of the transmission shaft 1606, a driving motor 1607 is also installed on the movable plate 11, and bevel gears meshed with the output shaft of the driving motor 1607 and the transmission shaft 1606 are respectively installed on the driving motor 1607, and the output end of the PCL controller 6 are electrically connected, and the forward or reverse running of the driving motor 1607 is controlled by editing instructions of the PCL controller 6.

In the above-described embodiments, the following embodiments are only preferred embodiments of the present invention, and are not the only embodiments, in order to enable the automatic control and the circuit operation principle of the present invention.

As shown in fig. 13, in the foregoing technical solution, the PCL controller is internally provided with an STM32RCT6 singlechip, where the STM32RCT6 singlechip is electrically connected with a display module (i.e., the display circuit in fig. 13), a key input module (i.e., the key control circuit in fig. 13), a programming interface module (i.e., the programming interface circuit in fig. 13), a storage module (i.e., the storage circuit in fig. 13), and a power management module 20 (i.e., the power management circuit in fig. 13) externally connected with the STM32RCT6 singlechip. The specific connection mode between the STM32RCT6 singlechip and each circuit is as follows: the PA8-10 pins of the STM32RCT6 singlechip are respectively connected with the 1-3 pins of the display circuit; the pins PA1 and PA4-7 of the STM32RCT6 singlechip are respectively connected with the pins 1-5 of the key control circuit; the NRST pin of the STM32RCT6 singlechip is connected with the D+ pins of two U5 in the programming interface circuit; PB13-15 pins of the STM32RCT6 singlechip are respectively connected with SDA, SCL, WP pins of U2 in the storage circuit; PB13-15 pin of STM32RCT6 singlechip; the power management module 20 selects an IP5306-CK charging motherboard, a VIN pin and a GND1 pin of the IP5306-CK charging motherboard are respectively connected with an anode and a cathode of the charging interface 7, a BAT pin and a GND2 pin of the IP5306-CK charging motherboard are respectively connected with an anode and a cathode of the lithium battery 19, a VOUT pin and a GND3 pin of the IP5306-CK charging motherboard are respectively connected with a VCC pin and a VSS pin of the STM32RCT6 singlechip, and a power button 8 (i.e., S0) is connected in series between the KEY pin and the GND3 pin to form a power management circuit (see fig. 13). And five wiring pins of PB3-7 of STM32RCT6 singlechip are connected with the second servo motor 12, the driving motor 1607, the micropump 21, the electromagnetic valve group 22 and the first servo motor 35 respectively.

The circuit principle designs of the display circuit, the key control circuit, the programming interface circuit and the storage circuit are common and can be used, so that the invention is not repeated here.

The method for extracting the bone marrow cells by adopting the full-automatic mouse bone marrow flushing device in the technical scheme comprises the following steps:

step 1: opening the turnover panel 4 to turn over the support platform 103, placing the femur and/or tibia of the stripped mouse on each pair of clips 36 in sequence using forceps, placing epiphyses at both ends of each femur or tibia between the arc-shaped blade 1602 and the blade groove 1601, and closing the turnover panel 4 on the outer case 1;

step 2: pressing the power button 8 activates the device and places the device on the ultra clean bench;

step 3: the driving motor 1607 starts and drives the arc blade 1602 to rotate according to the editing instruction of the PCL controller 6 to cut off the epiphysis at two ends of each femur or tibia, and the cutting operation instruction is completed;

step 4: the two second servo motors 12 start the common driving movable plate 11 to move upwards according to the editing instruction of the PCL controller 6, so that the flushing needle 26 is inserted into the femur or tibia to finish the alignment access operation instruction;

step 5: firstly, a pipeline switch provided with femur or tibia is opened by an electromagnetic valve group 22 according to an editing instruction of a PCL controller 6, then a micro pump 21 is started according to the editing instruction of the PCL controller 6, hanks buffer solution in a cleaning solution storage tank 23 is pumped into a marrow cavity, then two second servo motors 12 are started according to the editing instruction of the PCL controller 6 to jointly drive a movable plate 11 to move up and down circularly, meanwhile, a first servo motor 35 is started according to the editing instruction of the PCL controller 6, a collecting tank 30 and a collecting cavity 27 are matched with the upper and lower circularly of the movable plate 11, a mouse marrow suspension sequentially enters the collecting cavity 27 and a collecting box 5 through a spherical filter cover 28, and part of air in the telescopic hose 32 enters the collecting tank 30 in the telescopic process of the telescopic hose 32 in the whole upper and lower circularly moving process, and the air entering the telescopic hose 32 enters the cell flushing liquid in the collecting box 5 along the telescopic hose 32 to play a role of blowing;

step 6: after the step 5 is completed, firstly the micro pump 21 is closed according to the editing instruction of the PCL controller 6, then the electromagnetic valve group 22 closes all flushing channel switches according to the editing instruction of the PCL controller 6, and finally the two second servo motors 12 start the common driving movable plate 11 to move downwards according to the editing instruction of the PCL controller 6 so that the flushing needle 26 is completely separated from the bone marrow cavity of the femur or tibia, and the flushing operation instruction is completed;

step 7: the ultra-clean bench is opened, the device is taken out, the collecting box 5 is pulled out, and the cell flushing liquid is uniformly pumped into the container by using the dropper for standby.

Before the device is used, the parameters of the starting time and the sequence of the second servo motor, the driving motor, the micropump, the electromagnetic valve group and the first servo motor are firstly downloaded and transmitted through a programming interface module of the PCL controller 6 or manually edited and programmed through a key input module, and are stored by a storage module, and meanwhile, the related execution command parameters are displayed by a display module. After the power button 60s is started, the driving motor 1607 is arranged to rotate forward and backward for 15s respectively to cut epiphysis at two ends of femur and tibia; setting a second servo motor to rotate forward for 10s after the driving motor finishes 5s so that the bone marrow cavity moves upwards to the flushing needle opening, setting a corresponding channel opened by the electromagnetic valve group to be 300s after the second servo motor finishes 5s, and setting a micropump to start for 300s at the same time; after the electromagnetic valve group and the micropump are started for 5 seconds, the second servo motor and the first servo motor are synchronously arranged to rotate forward and backward for 150 seconds respectively, so that the femur and the tibia move up and down circularly, and the collecting cavity and the collecting groove move up and down circularly; after the operation of the micropump, the electromagnetic valve group, the second servo motor and the first servo motor is totally finished for 5 seconds, the second servo motor is arranged to reverse for 10 seconds, so that the marrow cavity moves downwards to be far away from the flushing needle opening, and the operation of programming parameter setting is finished, and the device can be used.

For the flexibility and convenience of practical use, the electromagnetic valve group can be manually edited through the key input module to control the opening and closing of different pipelines, so that the use of flushing liquid is effectively saved.

Of course, the above programming parameter setting is not the only implementation, and the related operation parameters can be temporarily and manually programmed through the key input module according to actual use.

On the basis of completing the cell flushing liquid prepared in the step 7, a 10 ml sterile centrifuge tube is used, 3ml of lymphocyte separation liquid special for animals is added, and then 1ml of evenly mixed marrow suspension (cell flushing liquid) is slowly dripped at a position about 1cm away from the liquid level of the lymphocyte separation liquid, so that an obvious interface is formed, and the upper liquid level and the lower liquid level are not mixed; then using an SC-3612 low-speed centrifuge for 2000 rpm for 20 minutes, carefully sucking the middle white membrane layer, diluting with 0.01mol/LPBS liquid with 7-10 times of volume, and then using the SC-3612 low-speed centrifuge for 1000 rpm for 10 minutes, and washing for 2-3 times; finally, 2ml of IMDM medium containing 10% fetal bovine serum, 100U/ml penicillin+100 ug/ml streptomycin was used to resuspend bone marrow cells in six well plates and cultured in a 37℃5% CO2 incubator.

Referring to fig. 15, by comparing the control pictures before and after flushing with the conventional manual flushing mode (fig. a) and the flushing mode (fig. b) of the device of the invention, we have no difficulty in seeing that the effect of cutting the incision of epiphysis and the bone marrow cavity permeability before and after flushing with the device of the invention is obviously better than that of the conventional manual flushing, namely, the incision regularity index of femur and tibia and the bone marrow cavity permeability.

The following table is a comparison of the number, status, activity and time of bone marrow hematopoietic stem cells obtained by 2 different extraction methods:

in combination with the chart and the attached figure 16, we can conclude that the number, the state and the activity of the bone marrow hematopoietic stem cells obtained by the novel device and the extraction method of the invention are better than those of the bone marrow hematopoietic stem cells obtained by the traditional manual method; and is significantly higher in use than conventional hand.

While the principal features and advantages of the present invention have been shown and described, it will be apparent to those skilled in the art that the detailed description of the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other embodiments without departing from the spirit or essential characteristics of the invention, and the inventive concept and design concept of the invention shall be equally included in the scope of the invention disclosed in the appended claims. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims (6)

1. The utility model provides a full automatization mouse marrow washing unit, is including shell body (1) be provided with handle (2) on the opposite both sides face of shell body (1) respectively, install apron (102), its characterized in that on its back: the top surface of the outer shell (1) is respectively provided with a PCL controller (6) and a power button (8), the outer shell (1) is respectively provided with a lithium battery (19), a power management module (20), a micro pump (21), an electromagnetic valve group (22) and a cleaning liquid storage tank (23), the output end of the power management module (20) is electrically connected with the lithium battery (19) and charges the lithium battery (19), the output end of the power management module (20) is electrically connected with the PCL controller (6) and supplies power to the PCL controller (6), the power button (8) is connected in series between the PCL controller (6) and the power management module (20) and is used for controlling the circuit on-off of the PCL controller (6), the power management module (20) is provided with a charging interface (7), the micro pump (21) and the electromagnetic valve group (22) are respectively connected with the output end of the PCL controller (6) through the power management module (20) to charge the lithium battery (19), and the micro pump (21) and the cleaning liquid storage tank (23) are respectively connected with the power supply through the power management module (20) through the power button and the electromagnetic valve group (21) to run through the micro pump (21), the outlet of micropump (21) is linked together through the import of pipeline and solenoid valve group (22), a plurality of exports of solenoid valve group (22) are respectively through the pipeline connection there are a plurality of buffer chamber pipes (25), in every all be provided with on buffer chamber pipe (25) wash needle (26) still correspond every wash needle (26) in shell body (1) be provided with collection chamber (27), every the bottom intercommunication of collection chamber (27) has runner pipe (29), runner pipe (29) all are through fixed plate (31) fixed connection on collecting vat (30), collecting vat (30) activity sets up on support frame (18) to provide power by first electric screw subassembly and realize upper and lower removal, support frame (18) are installed on base support (17), base support (17) are installed in shell body (1) the bottom of collecting vat (30) is connected with support frame (18) through flexible hose (32) to correspond on support frame (18) the bottom intercommunication has runner pipe (29), runner pipe (29) all are through fixed plate (31) fixed connection on collecting vat (30), and be provided with by first electric screw subassembly power and realize moving up and down on support frame (18), support frame (18) are installed on base support frame (17) are installed in shell body (1) and are provided with flexible box (32) and are provided with on the side face plate (4) and are provided with on the electric box (4) and are provided with the roll-over panel (4) and are provided with roll-over panel (4 on the top that is provided with roll-over panel (4) A pair of clamping pieces (36) and electric shearing assemblies (16) are arranged on the movable plate (11) corresponding to each flushing needle (26), each clamping piece (36) and each electric shearing assembly (16) are arranged on the movable plate (11) through a mounting bottom plate (14), each clamping piece (36) is commonly used for clamping the femur or the tibia of the same mouse, the corresponding two electric shearing assemblies (16) shear the epiphysis at two ends of the femur or the tibia, and the first electric screw assembly, the second electric screw assembly and the electric shearing assemblies (16) respectively perform operation and match to complete the shearing of the epiphysis, the movement of the femur and/or the tibia, the movement of the collecting cavity (27) and the movement of the collecting groove (30) through executing the editing instruction of the PCL controller (6);

the first electric screw rod assembly comprises a first servo motor (35) arranged on a supporting frame (18), a first screw rod (34) is arranged at the output end of the first servo motor (35), the other end of the first screw rod (34) is rotatably arranged on the supporting frame (18), a first screw hole plate (39) is arranged on the collecting tank (30) corresponding to the first screw rod (34), sliding rails (33) are respectively arranged at four corners of the supporting frame (18) corresponding to the collecting tank (30), and the first servo motor (35) is electrically connected with the output end of the PCL controller (6) and controls the forward or reverse operation of the first servo motor (35) through an editing instruction of the PCL controller (6);

the second electric screw rod assembly comprises two second servo motors (12) arranged on a turnover panel (4), each second servo motor (12) is provided with a second screw rod (13) at the output end, the other end of each second screw rod (13) is rotatably arranged on the turnover panel (4), at least two second screw hole plates (40) are arranged on the movable plate (11) corresponding to each second screw rod (13), two guide rail grooves (15) are arranged on the turnover panel (4), guide rails (37) are arranged on the movable plate (11) corresponding to the two guide rail grooves (15), and the second servo motors (12) are electrically connected with the output end of the PCL controller (6) and control the forward or reverse operation of the second servo motors (12) through editing instructions of the PCL controller (6);

each electric shear assembly (16) comprises an arc-shaped blade (1602) hinged on a mounting bottom plate (14), and a blade slot (1601) fixedly connected on the mounting bottom plate (14) corresponding to the arc-shaped blade (1602), wherein a plurality of arc-shaped blades (1602) positioned on the same level are hinged on a transmission rod (1603) together through a hinged connection sheet (1608), at least two limit sliding sleeves (38) are arranged on the movable plate (11) corresponding to each transmission rod (1603), a rack (1604) is fixedly connected on the transmission rod (1603), a transmission shaft (1606) is rotatably arranged on the movable plate (11), transmission gears (1605) meshed with the rack (1604) are respectively arranged at two ends of the transmission shaft (1606), a driving motor (1607) is further arranged on the movable plate (11), and conical gears meshed with the output shafts of the driving motor (1607) and the transmission shaft (1606) are respectively arranged on the movable plate (11), and the driving motor (1607) and the output end of a PCL controller (6) are electrically connected with the driving motor (1607) and controlled by a forward command or a reverse command (6);

a spherical filter cover (28) is arranged in each collecting cavity (27);

a supporting platform (103) is also arranged on the front surface of the outer shell (1), and the supporting platform (103) is used for supporting the overturned panel (4).

2. The fully automated mouse bone marrow flushing device of claim 1, wherein: the end part of the flushing needle (26) is communicated with a hollow ball (2601), and a plurality of flushing holes (2602) are formed in the hollow ball (2601).

3. The fully automated mouse bone marrow flushing device of claim 1, wherein: the spherical filter housing (28) is a 300 mesh screen for collecting the sheared epiphysis, thereby preventing the epiphysis from clogging into the flow tube (29) or into the collecting tank (30).

4. The fully automated mouse bone marrow flushing device of claim 1, wherein: the top surface of the outer shell (1) is also provided with a liquid injection port (101), a cap (3) is arranged on the liquid injection port (101), and a liquid injection connecting pipe (24) communicated with the liquid injection port (101) is arranged on the cleaning liquid storage tank (23).

5. A fully automated mouse bone marrow flushing device according to any one of claims 1 to 4, wherein: the outer side surface of the turnover panel (4) is provided with a handle (10) and a pressing lock catch (9), and the pressing lock catch (9) is used for temporarily locking the turnover panel (4) on the outer shell (1).

6. A method for bone marrow cell extraction using a fully automated mouse bone marrow washing device of claim 5, comprising the steps of:

step 1: opening the turnover panel (4) to enable the turnover panel to be turned over to the supporting platform (103), placing thighbone and/or tibia of a stripped mouse on each pair of clamping pieces (36) in sequence by using forceps, enabling epiphyses at two ends of each thighbone or tibia to be positioned between the arc-shaped blade (1602) and the cutting edge groove (1601), and closing the turnover panel (4) on the outer shell (1);

step 2: pressing a power button (8) to start the device and placing the device on an ultra-clean workbench;

step 3: the driving motor (1607) starts and drives the arc blade (1602) to rotate according to the editing instruction of the PCL controller (6) to cut off the epiphysis at two ends of each femur or tibia, and the cutting operation instruction is completed;

step 4: the two second servo motors (12) start the common driving movable plate (11) to move upwards according to the editing instruction of the PCL controller (6), so that the flushing needle (26) is inserted into the femur or tibia to finish the alignment access operation instruction;

step 5: firstly, an electromagnetic valve group (22) opens a pipeline switch provided with femur or tibia according to an editing instruction of a PCL controller (6), then a micro pump (21) is started according to the editing instruction of the PCL controller (6), hanks buffer solution in a cleaning liquid storage tank (23) is pumped into a marrow cavity, then two second servo motors (12) are started to jointly drive a movable plate (11) to move up and down circularly according to the editing instruction of the PCL controller (6), meanwhile, a first servo motor (35) is started according to the editing instruction of the PCL controller (6) to enable a collecting groove (30) and a collecting cavity (27) to move up and down circularly in cooperation with the movable plate (11), mouse marrow suspension sequentially enters the collecting cavity (27) and a collecting box (5) through a spherical filter cover (28), and in the whole up and down circular moving process, part of air in the telescopic hose (32) enters the collecting groove (30), and the air entering the telescopic hose enters into cells in the collecting box (5) along the telescopic hose (32) to play a role of blowing a dropper;

step 6: after the step 5 is completed, firstly, the micro pump (21) is closed according to the editing instruction of the PCL controller (6), then the electromagnetic valve group (22) is closed according to the editing instruction of the PCL controller (6), and finally, the two second servo motors (12) start the common driving movable plate (11) to move downwards according to the editing instruction of the PCL controller (6) so that the flushing needle (26) is completely separated from the bone marrow cavity of the femur or tibia, and the flushing operation instruction is completed;

step 7: the ultra-clean workbench is opened, the device is taken out, the collecting box (5) is pulled out, and the cell flushing liquid is uniformly pumped into the container by using the dropper to be reserved.

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